Riccardo Calafiore | Università degli Studi di Perugia (original) (raw)
Papers by Riccardo Calafiore
Biomaterials, 2016
Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degene... more Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration leading to impaired locomotion, respiratory failure and premature death. In DMD patients, inflammatory events secondary to dystrophin mutation play a major role in the progression of the pathology. Sertoli cells (SeC) have been largely used to protect xenogeneic engraftments or induce trophic effects thanks to their ability to secrete trophic, antiinflammatory, and immunomodulatory factors. Here we have purified SeC from specific pathogen-free (SPF)-certified neonatal pigs, and embedded them into clinical grade alginate microcapsules. We show that a single intraperitoneal injection of microencapsulated SPF SeC (SeC-MC) in an experimental model of DMD can rescue muscle morphology and performance in the absence of pharmacologic immunosuppressive treatments. Once i.p. injected, SeC-MC act as a drug delivery system that modulates the inflammatory response in muscle tissue, and upregulates the expression of the dystrophin paralogue, utrophin in muscles through systemic release of heregulin-β1, thus promoting sarcolemma stability. Analyses performed five months after single injection show high biocompatibility and long-term efficacy of SeC-MC. Our results might open new avenues for the treatment of patients with DMD and related diseases.
Xenotransplantation, 2015
Background: porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical stud... more Background: porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases because deliver molecules with trophic and antiinflammatory effects. In order to be employable for human long-term xenografts, pSCs products have to grant safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. Methods: SPF-certified neonatal pigs underwent testis excision under sterility. pSCs swere isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA), and examined in terms of viability and function [in terms of production of anti-mullerian hormone (AMH), inhibin B, transforming growth factor beta-1 (TFGbeta-1)]. After microencapsulation in barium-alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentration were evaluated over 0, 24 and 40 hours to determine pre-transplant storage conditions. Results: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior and after microencapsulation, maintained high function and viability up to 24 hours of storage. At 40 hours post encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and were stable for 4 months in mice with no evident side effects. Conclusions: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantion.
Gastroenterology, Jan 17, 2015
&... more & Aims: Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects. MSCs were isolated from bone marrow (BM-MSCs) of 4-6 week old C57BL/6, C57BL/6-GFP or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hrs later; histologic and flow cytometry analyses were performed. Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells and produced immune regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-β, arginase type II, CCL22, heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared to mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with numbers of CD45+ cells neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of inteferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis. BM- MSCs injected into mice with colitis do not localize to the intestine, but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis.
Trends in Biotechnology, 2004
Cell microencapsulation continues to hold significant promise for biotechnology and medicine. The... more Cell microencapsulation continues to hold significant promise for biotechnology and medicine. The controlled, and continuous, delivery of therapeutic products to the host by immunoisolated cells is a potentially cost-effective method to treat a wide range of diseases. Although there are several issues that need to be addressed, including capsule manufacture, properties and performance, in the past few years, a stepwise analysis on the essential obstacles and limitations has brought the whole technology closer to a realistic proposal for clinical application. This paper summarizes the current situation in the cell encapsulation field and discusses the main events that have occurred along the way.
Transplantation Proceedings, 2004
Neonatal pig pancreata could represent an ideal tissue resource for donor islets for transplantat... more Neonatal pig pancreata could represent an ideal tissue resource for donor islets for transplantation trials. Because functional islet beta-cells could derive from precursors situated in the ductal system, and neonatal animals are better suitable than adults for recovering such elements, we have examined whether isolated neonatal pancreatic ducts (NPD) could form insulin-producing cells. NPD, retrieved from the pancreas by collagenase digestion, were cultured for 2 weeks. A compact tissue monolayer detached by trypsin was re-incubated to form upon culture. The primary tissue monolayer was plated, yielding secondary monolayers that were supplemented in culture with the following factors: insulin transferrin selenium, niacinamide, keratinocyte growth factor, and high glucose, which promoted formation of islet cell-like clusters during 30 days of culture. Upon reaching 50 to 100 microm in diameter, the cell clusters were subjected to morphologic examination (assessment of viability by staining with ethidium bromide+fluorescein diacetate [EB+FD]; staining for insulin with diphenylthiocarbazone [DTZ]); DNA assay; insulin radioimmunoassay both in the basal state and after in vitro static incubation with high glucose; immunolabeling with anti-insulin fluorescent antibodies. Of the cell clusters, 80% were composed of viable cells that faintly showed DTZ staining. Basal insulin was 16.7 microU/mL, but no insulin response was elicited by stimulation with high glucose. Acid-ethanol extraction showed high insulin levels in the clusters. Finally, immunofluorescence for insulin was positive, indicating the presence of beta-cell-like committed elements. In conclusion, NPD may differentiate into insulin-producing cells, which are at a very early stage when the glucose-sensing apparatus is still immature.
Transplant Immunology, 2004
Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical ba... more Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical barrier to prevent islet graft (TX)-directed immune destruction. However, extent of the achieved immunoprotection will continue to be variable and unpredictable until the role of the individual mechanisms involved with TX-related inflammatory cell and immune reactivity are clarified. Macrophages (M) are believed to play a pivotal role in controlling the host/TX interaction and its consequences. We then have studied the effects of isolated rat M and their secretory products on allogeneic islets enveloped in variably sized and configured microcapsules, within in vitro mixed islet-M cocultures. In particular, we aimed to determine the sequence of immune or not immune specific cascade of early events that derive from such on interaction. One of the specific aims was to assess whether the membrane's physical intactness and conversely its even minimal rupture, along with the microcapsules' size (i.e., large vs. small) would significantly impact M reactivity and, thereby, the encapsulated islet viability and function. Special care was taken to evaluate extent of the elicited reactivity by meticulously monitoring cytokine, N2 derivative, and other proinflammatory protein curve profiles during the early M activation process. The study has preliminarily shown that, for equally formulated microcapsules, the capsular size and membrane's morphologic thoroughness are key to prevent M reactivity and possibly avoid the intracapsular islet cell damage. While elucidation of pathways involved with the encapsulated islet TX-directed host's responsiveness actually is in progress, it has clearly emerged that microcapsules should comply with well-defined physical properties and formulation specifications in order to obviate the primum movens of the inflammatory reaction process.
Pharmaceutical Research, 2010
Purpose To assess whether prevention of unexpected in vivo adverse inflammatory and immune respon... more Purpose To assess whether prevention of unexpected in vivo adverse inflammatory and immune responses to biohybrid organ grafts for the treatment of Type I Diabetes Mellitus (T1DM) is possible by superoxide dismutase and ketoprofen controlled release. Methods Superoxide dismutase and ketoprofen-loaded polyester microspheres were prepared by W/O/W and O/W methods, embodied into purified alginate-poly-L-ornithine-alginate microcapsules and intraperitoneally implanted into CD1 mice. The microspheres were characterized for morphology, size, encapsulation efficiency, enzyme activity and in vitro release. Purified alginate contaminants were assayed, and the obtained microcapsules were investigated for size and morphology before and after implantation over 30 days. Cell pericapsular overgrowth and expression were evaluated by optical microscopy and flow cytometry. Results Superoxide dismutase and ketoprofen sustained release reduced cell pericapsular overgrowth in comparison to the control. Superoxide dismutase release allowed preserving the microcapsules over 30 days. Ketoprofen-loaded microspheres showed some effect in the immediate post-grafting period. A higher macrophage and T-cell expression was observed for the control group. Conclusions Microspheres containing superoxide dismutase and ketoprofen may represent novel tools to limit or prevent unpredictable adverse in vivo response to alginate, thus contributing to improve cell transplantation success rates in T1DM treatment.
Journal of Experimental Medicine, 2009
Type I diabetes mellitus is caused by autoimmune destruction of pancreatic β cells, and effective... more Type I diabetes mellitus is caused by autoimmune destruction of pancreatic β cells, and effective treatment of the disease might require rescuing β cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional β cell or insulin therapy. The effect was associated with restoration ...
Human Reproduction, 2011
background: Spermatogenesis is a complex process where spermatogonial germ cells become spermatoz... more background: Spermatogenesis is a complex process where spermatogonial germ cells become spermatozoa with the indispensable support of Sertoli cells (SCs), which provide 'ad hoc' structural and nutritional support. Unfortunately, for most sperm dysfunctions, no therapies are yet available except assisted reproductive technologies (ART) that are based on the use of different culture media to preserve sperm in vitro. However, sperm culture is only possible for short periods of time, since long-term culture would invariably and irreversibly damage the cells with negative impact on their fertilization potential. methods: Fresh sperm cells (5 ml of 20 × 10 6 /ml) were co-cultured with SCs layers, derived from prepubertal pig testes or incubated in cell free SC medium or BWW (Biggers, Whitten and Whittingham) medium for 2, 4 or 7 days. Sperm viability, motility, mitochondrial status, DNA fragmentation, chromatin integrity, intracellular calcium and acrosome status were assessed after every co-culture or incubation time, but capacitation and induction of acrosome reaction (AR) with progesterone was only evaluated after 7 days. results: SCs layers derived from prepubertal pig testes (co-culture of sperm and SC feeder, CCSCF) were able to preserve normal sperm viability, motility and normal mitochondrial function, after 7 days of culture; CCSCF did not induce AR or hyperactivation of spermatozoa, keeping the sperm in a quiescent state for 7 days of culture. Nevertheless, the sperm were readily able to initiate AR after stimulation with progesterone. conclusions: CCSCF maintained good sperm viability and motility for 7 days. This approach could improve retention of sperm viability and motility during ART procedures and maintain sperm viability, during transfer between two distant Centres, avoiding the need for cryopreservation.
Biotechnology and Applied Biochemistry, 2004
Pancreatic-islet-cell transplantation may reverse hyperglycaemia in diabetic recipients that unde... more Pancreatic-islet-cell transplantation may reverse hyperglycaemia in diabetic recipients that undertake general pharmacological immunosuppression. A major challenge that remains is the need to avoid immunosuppression associated with the use of allogeneic or heterologous islet cells. In the present study we demonstrate the use of microencapsulation of cells using artificial biocompatible and permselective membranes prepared with alginic acid derivatives and polyamino acids. While characterization of the microcapsule constituent polymers continues to progress, other technical issues such as definition of the immunobarrier capacity, biocompatibility, size, shape and graft site have come into sharper focus. Assessment of microcapsules properties, in order to establish possible guidelines for fabrication of reproducible membranes, and results from both in vitro functional testing, and in vivo encapsulated-islet-transplant outcome in several animal models of diabetes are reported.
Biomaterials, 2003
Great advances in cell transplantation have been made, including the recent, remarkable success i... more Great advances in cell transplantation have been made, including the recent, remarkable success in pancreatic islet transplantation for the treatment of type 1 diabetes mellitus. Unfortunately, the transplanted cells are very susceptible to oxidative stress that cause severe damage to either allo-or xenogeneic islets upon graft in diabetic patients. Consequently, the transplanted islet functional life span is significantly shortened. The aim of this study was to examine the possible effects of antioxidants on in vitro cultured adult rat islets, and to evaluate the effects of a prolonged-release formulation, in form of cellulose acetate (CA) microspheres, on Vitamin D 3 activity. Isolated rat islets, both free and entrapped in microspheres were treated with Vitamin D 3. The effects of the vitamin were studied at 3, 6 and 9 days of in vitro cell culture. According to insulin secretory patterns, treatment with Vitamin D 3 of both free and CA entrapped microspheres, increased the insulin output as compared to untreated controls. Such positive effects were confirmed under islet static incubation with glucose at day 6. These results suggest that pancreatic islets can be advantageously treated with anti-oxidising vitamins before implantation, and speculatively, with the help of special delivery systems, throughout the islet cell life span, in the post-transplant time period.
Acta Biomaterialia, 2011
A method for the production of engineered alginate-based microparticles, containing extracellular... more A method for the production of engineered alginate-based microparticles, containing extracellular matrix and neonatal porcine Sertoli cells (SCs), is described. As a source for extracellular matrix, a powder form of isolated and purified urinary bladder matrix (UBM) was employed. We demonstrated that the incorporation of UBM does not significantly alter the morphological and dimensional characteristics of the microparticles. The alginate microparticles were used for SC encapsulation as an immunoprotective barrier for transplant purposes, while the co-entrapped UBM promoted retention of cell viability and function. These engineered microparticles could represent a novel approach to enhancing immunological acceptance and increasing the functional lifespan of the entrapped cells for cell/tissue engineering applications. In this respect, it is noteworthy that isolated neonatal porcine SCs, administered alone in highly biocompatible microparticles, led to diabetes prevention and reversion in nonobese diabetic (NOD) mice.
American journal of stem cells, 2015
In spite of intense research, over the past 2-3 decades, targeted to validating methods for the c... more In spite of intense research, over the past 2-3 decades, targeted to validating methods for the cure of T1D, based on cell substitution therapy in the place of exogenously administered insulin injections, achievement of the final goal continues to remain out of reach. In fact, aside of very limited clinical success of the few clinical trials of pancreatic islet cell transplantation in totally immunosuppressed patients with T1D, the vast majority of these diabetic patients invariably is insulin-dependent. New advances for cell and molecular therapy for T1D, including use of stem cells, are reviewed and discussed in an attempt to clearly establish where we are and where are we may go for the final cure for T1DM.
Biomaterials, 2015
Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degene... more Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration leading to impaired locomotion, respiratory failure and premature death. In DMD patients, inflammatory events secondary to dystrophin mutation play a major role in the progression of the pathology. Sertoli cells (SeC) have been largely used to protect xenogeneic engraftments or induce trophic effects thanks to their ability to secrete trophic, antiinflammatory, and immunomodulatory factors. Here we have purified SeC from specific pathogen-free (SPF)-certified neonatal pigs, and embedded them into clinical grade alginate microcapsules. We show that a single intraperitoneal injection of microencapsulated SPF SeC (SeC-MC) in an experimental model of DMD can rescue muscle morphology and performance in the absence of pharmacologic immunosuppressive treatments. Once i.p. injected, SeC-MC act as a drug delivery system that modulates the inflammatory response in muscle tissue, and upregulates the expression of the dystrophin paralogue, utrophin in muscles through systemic release of heregulin-β1, thus promoting sarcolemma stability. Analyses performed five months after single injection show high biocompatibility and long-term efficacy of SeC-MC. Our results might open new avenues for the treatment of patients with DMD and related diseases.
Transactions of the Association of American Physicians
Biomaterials, 2016
Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degene... more Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration leading to impaired locomotion, respiratory failure and premature death. In DMD patients, inflammatory events secondary to dystrophin mutation play a major role in the progression of the pathology. Sertoli cells (SeC) have been largely used to protect xenogeneic engraftments or induce trophic effects thanks to their ability to secrete trophic, antiinflammatory, and immunomodulatory factors. Here we have purified SeC from specific pathogen-free (SPF)-certified neonatal pigs, and embedded them into clinical grade alginate microcapsules. We show that a single intraperitoneal injection of microencapsulated SPF SeC (SeC-MC) in an experimental model of DMD can rescue muscle morphology and performance in the absence of pharmacologic immunosuppressive treatments. Once i.p. injected, SeC-MC act as a drug delivery system that modulates the inflammatory response in muscle tissue, and upregulates the expression of the dystrophin paralogue, utrophin in muscles through systemic release of heregulin-β1, thus promoting sarcolemma stability. Analyses performed five months after single injection show high biocompatibility and long-term efficacy of SeC-MC. Our results might open new avenues for the treatment of patients with DMD and related diseases.
Xenotransplantation, 2015
Background: porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical stud... more Background: porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases because deliver molecules with trophic and antiinflammatory effects. In order to be employable for human long-term xenografts, pSCs products have to grant safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. Methods: SPF-certified neonatal pigs underwent testis excision under sterility. pSCs swere isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA), and examined in terms of viability and function [in terms of production of anti-mullerian hormone (AMH), inhibin B, transforming growth factor beta-1 (TFGbeta-1)]. After microencapsulation in barium-alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentration were evaluated over 0, 24 and 40 hours to determine pre-transplant storage conditions. Results: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior and after microencapsulation, maintained high function and viability up to 24 hours of storage. At 40 hours post encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and were stable for 4 months in mice with no evident side effects. Conclusions: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantion.
Gastroenterology, Jan 17, 2015
&... more & Aims: Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects. MSCs were isolated from bone marrow (BM-MSCs) of 4-6 week old C57BL/6, C57BL/6-GFP or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hrs later; histologic and flow cytometry analyses were performed. Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells and produced immune regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-β, arginase type II, CCL22, heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared to mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with numbers of CD45+ cells neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of inteferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis. BM- MSCs injected into mice with colitis do not localize to the intestine, but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis.
Trends in Biotechnology, 2004
Cell microencapsulation continues to hold significant promise for biotechnology and medicine. The... more Cell microencapsulation continues to hold significant promise for biotechnology and medicine. The controlled, and continuous, delivery of therapeutic products to the host by immunoisolated cells is a potentially cost-effective method to treat a wide range of diseases. Although there are several issues that need to be addressed, including capsule manufacture, properties and performance, in the past few years, a stepwise analysis on the essential obstacles and limitations has brought the whole technology closer to a realistic proposal for clinical application. This paper summarizes the current situation in the cell encapsulation field and discusses the main events that have occurred along the way.
Transplantation Proceedings, 2004
Neonatal pig pancreata could represent an ideal tissue resource for donor islets for transplantat... more Neonatal pig pancreata could represent an ideal tissue resource for donor islets for transplantation trials. Because functional islet beta-cells could derive from precursors situated in the ductal system, and neonatal animals are better suitable than adults for recovering such elements, we have examined whether isolated neonatal pancreatic ducts (NPD) could form insulin-producing cells. NPD, retrieved from the pancreas by collagenase digestion, were cultured for 2 weeks. A compact tissue monolayer detached by trypsin was re-incubated to form upon culture. The primary tissue monolayer was plated, yielding secondary monolayers that were supplemented in culture with the following factors: insulin transferrin selenium, niacinamide, keratinocyte growth factor, and high glucose, which promoted formation of islet cell-like clusters during 30 days of culture. Upon reaching 50 to 100 microm in diameter, the cell clusters were subjected to morphologic examination (assessment of viability by staining with ethidium bromide+fluorescein diacetate [EB+FD]; staining for insulin with diphenylthiocarbazone [DTZ]); DNA assay; insulin radioimmunoassay both in the basal state and after in vitro static incubation with high glucose; immunolabeling with anti-insulin fluorescent antibodies. Of the cell clusters, 80% were composed of viable cells that faintly showed DTZ staining. Basal insulin was 16.7 microU/mL, but no insulin response was elicited by stimulation with high glucose. Acid-ethanol extraction showed high insulin levels in the clusters. Finally, immunofluorescence for insulin was positive, indicating the presence of beta-cell-like committed elements. In conclusion, NPD may differentiate into insulin-producing cells, which are at a very early stage when the glucose-sensing apparatus is still immature.
Transplant Immunology, 2004
Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical ba... more Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical barrier to prevent islet graft (TX)-directed immune destruction. However, extent of the achieved immunoprotection will continue to be variable and unpredictable until the role of the individual mechanisms involved with TX-related inflammatory cell and immune reactivity are clarified. Macrophages (M) are believed to play a pivotal role in controlling the host/TX interaction and its consequences. We then have studied the effects of isolated rat M and their secretory products on allogeneic islets enveloped in variably sized and configured microcapsules, within in vitro mixed islet-M cocultures. In particular, we aimed to determine the sequence of immune or not immune specific cascade of early events that derive from such on interaction. One of the specific aims was to assess whether the membrane's physical intactness and conversely its even minimal rupture, along with the microcapsules' size (i.e., large vs. small) would significantly impact M reactivity and, thereby, the encapsulated islet viability and function. Special care was taken to evaluate extent of the elicited reactivity by meticulously monitoring cytokine, N2 derivative, and other proinflammatory protein curve profiles during the early M activation process. The study has preliminarily shown that, for equally formulated microcapsules, the capsular size and membrane's morphologic thoroughness are key to prevent M reactivity and possibly avoid the intracapsular islet cell damage. While elucidation of pathways involved with the encapsulated islet TX-directed host's responsiveness actually is in progress, it has clearly emerged that microcapsules should comply with well-defined physical properties and formulation specifications in order to obviate the primum movens of the inflammatory reaction process.
Pharmaceutical Research, 2010
Purpose To assess whether prevention of unexpected in vivo adverse inflammatory and immune respon... more Purpose To assess whether prevention of unexpected in vivo adverse inflammatory and immune responses to biohybrid organ grafts for the treatment of Type I Diabetes Mellitus (T1DM) is possible by superoxide dismutase and ketoprofen controlled release. Methods Superoxide dismutase and ketoprofen-loaded polyester microspheres were prepared by W/O/W and O/W methods, embodied into purified alginate-poly-L-ornithine-alginate microcapsules and intraperitoneally implanted into CD1 mice. The microspheres were characterized for morphology, size, encapsulation efficiency, enzyme activity and in vitro release. Purified alginate contaminants were assayed, and the obtained microcapsules were investigated for size and morphology before and after implantation over 30 days. Cell pericapsular overgrowth and expression were evaluated by optical microscopy and flow cytometry. Results Superoxide dismutase and ketoprofen sustained release reduced cell pericapsular overgrowth in comparison to the control. Superoxide dismutase release allowed preserving the microcapsules over 30 days. Ketoprofen-loaded microspheres showed some effect in the immediate post-grafting period. A higher macrophage and T-cell expression was observed for the control group. Conclusions Microspheres containing superoxide dismutase and ketoprofen may represent novel tools to limit or prevent unpredictable adverse in vivo response to alginate, thus contributing to improve cell transplantation success rates in T1DM treatment.
Journal of Experimental Medicine, 2009
Type I diabetes mellitus is caused by autoimmune destruction of pancreatic β cells, and effective... more Type I diabetes mellitus is caused by autoimmune destruction of pancreatic β cells, and effective treatment of the disease might require rescuing β cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional β cell or insulin therapy. The effect was associated with restoration ...
Human Reproduction, 2011
background: Spermatogenesis is a complex process where spermatogonial germ cells become spermatoz... more background: Spermatogenesis is a complex process where spermatogonial germ cells become spermatozoa with the indispensable support of Sertoli cells (SCs), which provide 'ad hoc' structural and nutritional support. Unfortunately, for most sperm dysfunctions, no therapies are yet available except assisted reproductive technologies (ART) that are based on the use of different culture media to preserve sperm in vitro. However, sperm culture is only possible for short periods of time, since long-term culture would invariably and irreversibly damage the cells with negative impact on their fertilization potential. methods: Fresh sperm cells (5 ml of 20 × 10 6 /ml) were co-cultured with SCs layers, derived from prepubertal pig testes or incubated in cell free SC medium or BWW (Biggers, Whitten and Whittingham) medium for 2, 4 or 7 days. Sperm viability, motility, mitochondrial status, DNA fragmentation, chromatin integrity, intracellular calcium and acrosome status were assessed after every co-culture or incubation time, but capacitation and induction of acrosome reaction (AR) with progesterone was only evaluated after 7 days. results: SCs layers derived from prepubertal pig testes (co-culture of sperm and SC feeder, CCSCF) were able to preserve normal sperm viability, motility and normal mitochondrial function, after 7 days of culture; CCSCF did not induce AR or hyperactivation of spermatozoa, keeping the sperm in a quiescent state for 7 days of culture. Nevertheless, the sperm were readily able to initiate AR after stimulation with progesterone. conclusions: CCSCF maintained good sperm viability and motility for 7 days. This approach could improve retention of sperm viability and motility during ART procedures and maintain sperm viability, during transfer between two distant Centres, avoiding the need for cryopreservation.
Biotechnology and Applied Biochemistry, 2004
Pancreatic-islet-cell transplantation may reverse hyperglycaemia in diabetic recipients that unde... more Pancreatic-islet-cell transplantation may reverse hyperglycaemia in diabetic recipients that undertake general pharmacological immunosuppression. A major challenge that remains is the need to avoid immunosuppression associated with the use of allogeneic or heterologous islet cells. In the present study we demonstrate the use of microencapsulation of cells using artificial biocompatible and permselective membranes prepared with alginic acid derivatives and polyamino acids. While characterization of the microcapsule constituent polymers continues to progress, other technical issues such as definition of the immunobarrier capacity, biocompatibility, size, shape and graft site have come into sharper focus. Assessment of microcapsules properties, in order to establish possible guidelines for fabrication of reproducible membranes, and results from both in vitro functional testing, and in vivo encapsulated-islet-transplant outcome in several animal models of diabetes are reported.
Biomaterials, 2003
Great advances in cell transplantation have been made, including the recent, remarkable success i... more Great advances in cell transplantation have been made, including the recent, remarkable success in pancreatic islet transplantation for the treatment of type 1 diabetes mellitus. Unfortunately, the transplanted cells are very susceptible to oxidative stress that cause severe damage to either allo-or xenogeneic islets upon graft in diabetic patients. Consequently, the transplanted islet functional life span is significantly shortened. The aim of this study was to examine the possible effects of antioxidants on in vitro cultured adult rat islets, and to evaluate the effects of a prolonged-release formulation, in form of cellulose acetate (CA) microspheres, on Vitamin D 3 activity. Isolated rat islets, both free and entrapped in microspheres were treated with Vitamin D 3. The effects of the vitamin were studied at 3, 6 and 9 days of in vitro cell culture. According to insulin secretory patterns, treatment with Vitamin D 3 of both free and CA entrapped microspheres, increased the insulin output as compared to untreated controls. Such positive effects were confirmed under islet static incubation with glucose at day 6. These results suggest that pancreatic islets can be advantageously treated with anti-oxidising vitamins before implantation, and speculatively, with the help of special delivery systems, throughout the islet cell life span, in the post-transplant time period.
Acta Biomaterialia, 2011
A method for the production of engineered alginate-based microparticles, containing extracellular... more A method for the production of engineered alginate-based microparticles, containing extracellular matrix and neonatal porcine Sertoli cells (SCs), is described. As a source for extracellular matrix, a powder form of isolated and purified urinary bladder matrix (UBM) was employed. We demonstrated that the incorporation of UBM does not significantly alter the morphological and dimensional characteristics of the microparticles. The alginate microparticles were used for SC encapsulation as an immunoprotective barrier for transplant purposes, while the co-entrapped UBM promoted retention of cell viability and function. These engineered microparticles could represent a novel approach to enhancing immunological acceptance and increasing the functional lifespan of the entrapped cells for cell/tissue engineering applications. In this respect, it is noteworthy that isolated neonatal porcine SCs, administered alone in highly biocompatible microparticles, led to diabetes prevention and reversion in nonobese diabetic (NOD) mice.
American journal of stem cells, 2015
In spite of intense research, over the past 2-3 decades, targeted to validating methods for the c... more In spite of intense research, over the past 2-3 decades, targeted to validating methods for the cure of T1D, based on cell substitution therapy in the place of exogenously administered insulin injections, achievement of the final goal continues to remain out of reach. In fact, aside of very limited clinical success of the few clinical trials of pancreatic islet cell transplantation in totally immunosuppressed patients with T1D, the vast majority of these diabetic patients invariably is insulin-dependent. New advances for cell and molecular therapy for T1D, including use of stem cells, are reviewed and discussed in an attempt to clearly establish where we are and where are we may go for the final cure for T1DM.
Biomaterials, 2015
Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degene... more Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration leading to impaired locomotion, respiratory failure and premature death. In DMD patients, inflammatory events secondary to dystrophin mutation play a major role in the progression of the pathology. Sertoli cells (SeC) have been largely used to protect xenogeneic engraftments or induce trophic effects thanks to their ability to secrete trophic, antiinflammatory, and immunomodulatory factors. Here we have purified SeC from specific pathogen-free (SPF)-certified neonatal pigs, and embedded them into clinical grade alginate microcapsules. We show that a single intraperitoneal injection of microencapsulated SPF SeC (SeC-MC) in an experimental model of DMD can rescue muscle morphology and performance in the absence of pharmacologic immunosuppressive treatments. Once i.p. injected, SeC-MC act as a drug delivery system that modulates the inflammatory response in muscle tissue, and upregulates the expression of the dystrophin paralogue, utrophin in muscles through systemic release of heregulin-β1, thus promoting sarcolemma stability. Analyses performed five months after single injection show high biocompatibility and long-term efficacy of SeC-MC. Our results might open new avenues for the treatment of patients with DMD and related diseases.
Transactions of the Association of American Physicians